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Performance of fluorescent dyes with LEADseeker Multimodality Imaging System

Alexa Fluor 488, 532, 546, 555, and 647 (Molecular Probes)

BODIPY-TMR and BODIPY-650/665 (Molecular Probes)

Fluorescein and TAMRA™ (Molecular Probes)


Protocol
All of the fluorescent dye reagents were dissolved from powder form into DMSO at approximately 10 mg/ml. Subsequently, each dye was diluted in buffer (PBS containing 0.01% [v/v] Brij™-35) to produce solutions for determining concentration. In all cases, we determined concentration by measuring absorbance (at the absorption maxima for the dye) against a buffer-only reference and by applying the Beer Lambert equation:

i.e. c = O.D. / εl where:
c = concentration
O.D. = absorption value determined
ε = extinction coefficient of the relevant fluorescent dye

Following determination of concentration, each dye was diluted further in buffer to a top working concentration of 6.4 nM. It was then subjected to doubling dilution in buffer to produce a low concentration of 0.00625 nM. Replicates of each dilution and buffer-only reference were added (as a total volume of 50 µl per well) to a 384-well microplate and imaged (using appropriate optical components) on LEADseeker Multimodality Imaging System in either fluorescence intensity or fluorescence polarization mode for a maximum of 10 s coincidence averaging counting.


Results
Following imaging, we determined the mean value of the signal and standard deviation at each concentration for data obtained from each of the replicates in fluorescence intensity mode. LLD was then determined as the lowest dye concentration with an observed intensity value greater than that of the buffer + 3 SD of the buffer-only wells. Table 1 shows the dat
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